Principal Leader

Anup Kollanoor Johny

Department

Department of Animal Science

Funding Awarded

2018 Fiscal Year: $50,000

2019 Fiscal Year: $39,342

The Problem

Minnesota ranks no. 1 in the production of turkeys with 45 million turkeys produced in 2014. S. Heidelberg, one of the most invasive of all Salmonella serotypes, has surfaced to importance causing significant economic loss to poultry industry, including turkeys.

Background

MDR Salmonella Heidelberg – an emerging problem of human health concern from turkeys: The USA is the world’s largest producer and exporter of turkeys and turkey products (USDA-ERS, 2016). Minnesota ranks no. 1 in turkey production in the USA and exports >$90 million worth turkey products to various countries (MDA, 2015). However, contamination of turkey products with Salmonella has resulted in product recalls in the past, causing significant economic losses to the industry (CDC, 2011a, b). Although industry mitigation efforts have significantly reduced the total numbers of Salmonella isolated from turkey products over the years, the recent NARMS report indicate an increase in the isolation of MDR Salmonella clones (46.7%) (NARMS, 2016). Historically, S. Heidelberg

has been one of the common Salmonella associated with poultry, including turkeys (Jackson et al., 2013). S. Heidelberg has been ranked seventh most common etiological agents of human salmonellosis in the USA (CDC, 2013 a, b, c) and is an emerging threat to the U.S. food supply due to high ABR potential (Routh et al., 2015; Folster et al., 2012a, b). Turkeys can carry MDR Salmonella in their ceca and can serve as critical sources for pathogen contamination of turkey products. In addition to birds, there are multiple sources for Salmonella on farms including bedding, drinkers, feed, box liners, yolk sac, ceca, feed shipments, and air (Nayak et al., 2003; Hoover., 1997) making the situation difficult to tackle. With the FDA’s final rule to phase out antibiotics from the production agriculture, A2A interventions that reduce the load of Salmonella, including MDR clones, are urgently required. Most importantly, the A2A interventions that have significant promise should be studied at applied and mechanistic levels for reliability and consistency like antibiotics, which will be the immediate outcome of the project. Ongoing efforts at UMN to control MDR S. Heidelberg using A2A interventions (2015-2016): Due to multiple sources of Salmonella on farms, no single approach effectively eliminates the pathogen. Keeping this in perspective, we envisioned the potential impact of a “combination approach” to control Salmonella in turkeys. Currently, we are comparing the efficacy of a newly introduced Salmonella vaccine in turkeys (Megan Vac Egg), a mannanoligosaccharide (MOS) - based prebiotic, and a DFM mix of turkey origin (Lactobacillus ingluviei and L. salivarius) independently, and in combination against MDR S. Heidelberg in turkeys. Results from the recently completed MTRPC project indicates that the combination of the tested prebiotic, DFM mix, and the vaccination performed significantly better than the independent interventions in turkey poults. In addition, we were successful in developing a challenge dose-response model in adult turkeys with the ongoing RR funds. This progress has led us to further team up for investigating the mechanistic roles played by the DFM mix, MOS, and the vaccination in the interacting gut environment of turkeys where potential changes in the microbiome, metabolome, and genome (virulence and ABR) exist. We have obtained samples from the MTRPC and the RR projects to do these investigations. More samples will be obtained when the ongoing RR project is completed (completion in June, 2017). Genomic analysis of virulence and ABR gene abundance in the gut: Several zoonotic pathogens have already filled their quivers with deadly arrows of multidrug resistance. Based on the MTRPC data, it is highly likely that A2A interventions could down-regulate the critical genes responsible for virulence and ABR in MDR S. Heidelberg. If this is the case, we expect to quantify changes in the virulence of the input (public health isolate before challenge) and output (recovered from turkeys after challenge) isolates using real-time quantitative polymerase chain reaction (RT-qPCR). The abundance of ABR genes in the microbial community will be assessed using highthroughput sequencing (HTS) - based metagenomic analysis. The HTS and RT-qPCR are strong tools to determine the effects of A2A interventions on MDR. S. Heidelberg virulence and ABR gene abundance. Our study will uniquely determine the alleviating effects of A2A interventions during pathogen challenge from the ABR perspective. Microbiome analysis to study pathogen-induced microbial shifts and effects of A2A interventions in the turkey gut: in the recent years, deciphering the poultry microbiome has been a major focus to improve production. Like humans, the poultry gastrointestinal tract is composed of complex microbial community linked to essential metabolic and immune functions, production of vitamins, and maintenance of gut health (Ballou et al., 2016; Pauwels et al., 2015). Several recent reports have indicated the potential of microbiome studies to explicitly understand the origins, progression, and control of infectious agents by effectively modulating the microbiome, since control of pathogens for product safety is economically important in production agriculture (Thibodeau et al., 2015; Oakley et al., 2014). However, there are no mechanistic studies exploring A2A interventions or their combinations on the turkey microbiome under pathogen challenge. This critical step will identify ways to modulate the turkey microbiota before pathogen colonization as a mitigation practice. Information on the effects of pathogen challenge on bacterial community structure (basic) and the community structure that is most susceptible to infection and with different A2A supplementation could be a valuable information to the turkey industry for devising appropriate mitigation methods. Metabolome analysis to explore chemical interactions in the turkey gut: Bacteria – host interactions at the intestinal interface could potentially result in changes in the chemical signature of the gut (Cevallos-Cevallos et al., 2011a). The growth of pathogenic bacteria in the gut could result in variations in the metabolic profile of the intestinal macrocosm; we can estimate this change using high throughput metabolomics (Cevallos-Cevallos et al., 2011b). Also, small molecules play important roles in the lifestyle of all organisms, including endocrine signaling, microbial communication, and metabolic interrelationships as in a microbial consortium where the release of secondary

metabolites with several biological functions would result (Sekirov et al., 2010), including endogenous antimicrobials (Antunes et al., 2011). This crucial step will obtain the snapshot of the chemical composition of the turkey cecal environment after the A2A treatments individually and in combination, in relation to MDR S. Heidelberg challenge. Once we have the microbiome and metabolome data sets, we would use this data to study the relationship between the normal microbial community and the chemical signature, and the shifts in this relationship structure in response to challenge and A2A treatments, which is innovative. Short- and long- term benefits of the project: The short-term benefits of the project will be to mechanistically validate the efficacy of multiple A2A interventions to control MDR S. Heidelberg colonization in turkeys. In addition, the information will be disseminated to the turkey producers in Minnesota. The long-term benefit of the project will be to prevent product recalls due to Salmonella contamination of turkey products and develop A2A-based mitigation methods helpful for the turkey producers in Minnesota considering the phase out of antibiotics, and ABR in bacteria.

Objectives

The specific goal of this integrated (research/extension), multi-college/department (CFANS/ANSC, CBS/CMSP, CVM/VBS) proposal is to understand the mechanisms underlying the efficacy of alternatives to antibiotic (A2A) interventions in combination against multidrug resistant (MDR) S. Heidelberg from the genomic-, microbiome and metabolome- perspectives. Results from the recently completed Minnesota Turkey Research and Promotion Council (MTRPC) project and the dose-response study results from the ongoing RR project indicate the efficacy of combining A2A interventions as a promising approach to control MDR Salmonella in turkeys. This renewal request is specifically made for the genomic-, microbiome-, and metabolome- analyses of samples collected from the MTRPC project that determined the efficacy of A2A interventions against S. Heidelberg in turkey poults, and the ongoing RR project that determines the efficacy of A2A interventions against S. Heidelberg in adult turkey hens. Our specific objectives are:

To understand the combined effect of vaccination, Direct Fed Microbials (DFM) of turkey origin, and prebiotics on MDR S. Heidelberg in turkeys mechanistically, using genomic analysis of virulence and antibiotic resistance (ABR) genes, and microbiome- and metabolome- analyses.

To disseminate project results (applied information from the MTRPC and ongoing RR projects, and mechanistic information from the proposed renewal) to turkey growers and public.